Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility

Abstract Enteroids are miniature self-organising three-dimensional (3D) tissue cultures which replicate much of the complexity of the intestinal epithelium. We recently developed an apical-out leukocyte-containing chicken enteroid model providing a novel physiologically relevant in vitro tool to explore host–pathogen interactions in the avian gut. However, the replicate consistency and culture stability have not yet been fully explored at the transcript level. In addition, causes for the inability to passage apical-out enteroids were not determined. Here we report the transcriptional profiling of chicken embryonic intestinal villi and chicken enteroid cultures using bulk RNA-seq. Comparison of the transcriptomes of biological and technical replicate enteroid cultures confirmed their high level of reproducibility. Detailed analysis of cell subpopulation and function markers revealed that the mature enteroids differentiate from late embryonic intestinal villi to recapitulate many digestive, immune and gut-barrier functions present in the avian intestine. These transcriptomic results demonstrate that the chicken enteroid cultures are highly reproducible, and within the first week of culture they morphologically mature to appear similar to the in vivo intestine, therefore representing a physiologically-relevant in vitro model of the chicken intestine.